EP2205251A1 - Procédé pour amplifier des cellules cardiaques souches in vitro et in vivo - Google Patents

Procédé pour amplifier des cellules cardiaques souches in vitro et in vivo

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Publication number
EP2205251A1
EP2205251A1 EP08835478A EP08835478A EP2205251A1 EP 2205251 A1 EP2205251 A1 EP 2205251A1 EP 08835478 A EP08835478 A EP 08835478A EP 08835478 A EP08835478 A EP 08835478A EP 2205251 A1 EP2205251 A1 EP 2205251A1
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Prior art keywords
cells
stem cells
cardiac
cell
mesenchymal stem
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EP08835478A
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German (de)
English (en)
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EP2205251B1 (fr
EP2205251A4 (fr
Inventor
Joshua M. Hare
Konstantinos Chatzistergos
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VESTION INC.
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University of Miami
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells

Definitions

  • Embodiments of the invention relate to stem cell compositions and methods of tissue repair, and amplifying cardiac stem cells in vivo and in vitro.
  • MSCs Mesenchymal stem cells
  • MI myocardial infarction
  • This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.
  • This invention addresses a major limitation in the cardiac cell therapy area. This limitation involves using cardiac stem cells to treat patients with heart disease. Cardiac stem cells, while highly promising as a therapy, require a tissue biopsy and a long period of growth in the laboratory. MSCs can be used to accelerate the growth of CSCs in vitro or can be used to amplify CSCs in vivo, thereby eliminating the need for the tissue biopsy.
  • a method of treating heart disease and heart disorders in a patient comprises isolating stem cells from a patient or donor; purifying the stem cells and obtaining mesenchymal stem cells; administering to a patient's cardiac tissue, mesenchymal stem cells in a concentration effective to repair damaged cardiac tissue; and, treating heart disease and heart disorders.
  • the mesenchymal stem cells are autologous or donor derived.
  • the mesenchymal stem cells differentiate into multi- lineages.
  • the mesenchymal stem cells differentiate into at least one lineage of cardiac cells; more preferably, the mesenchymal stem cells differentiate into at least two lineages of cardiac cells; more preferably, the mesenchymal stem cells differentiate into three lineages of cardiac cells.
  • the lineages of cardiac cells are indentified by at least one marker comprising cardiac transcription factor GATA-4; endothelial cell markers Factor VIII and KDR; vascular smooth muscle marker ⁇ -smooth muscle actin; or cardiomyocyte marker ⁇ -sarcomeric actinin.
  • the stem cells are obtained from bone marrow, circulation or tissues and organs.
  • the mesenchymal stem cells isolated from adult bone marrow cells.
  • the mesenchymal cells recruit endogenous cardiac stem cells, reconstitute myocardial stem cell niches and accelerate endogenous cell differentiation into myocytes.
  • the endogenous cardiac stem cells are indentified by at least one marker comprising connexin-43, N-cadherin, c-kit pos , CD3 neg , CD14 neg and CD68 neg .
  • cardiac stem cells are autologous or donor derived.
  • the mesenchymal stem cell factors are administered to the patient.
  • a method of recruiting endogenous cardiac stem cells to damaged heart tissue comprises administering to the damaged heart tissue, purified mesenchymal stem cells; and, recruiting endogenous cardiac stem cells.
  • the mesenchymal stem cells are purified from adult bone marrow.
  • the mesenchymal stem cells are autologous, heterologous, syngeneic, allogeneic or xenogeneic.
  • the damage to heart tissue can be from any source or cause and comprises disease, physical damage, chemical damage, surgery, transplantation, or congenital defects.
  • the method of inducing and/or accelerating cardiac stem cell proliferation comprises isolating mesenchymal stem cells; co-culturing mesenchymal stem cells and cardiac stem cells in a concentration sufficient to induce and/or accelerate cardiac stem cells proliferation.
  • the cardiac stem cells differentiate into cardiac cells expressing at least one of MDRl or GATA-4.
  • the cardiac stem cells are derived from an autologous or histocompatible tissue biopsy.
  • the isolated the mesenchymal cells are administered to a patient.
  • the mesenchymal cells and cardiac stem cells are autologous, heterologous, syngeneic, allogeneic or xenogeneic.
  • the mesenchymal stem cells and cardiac stem cells are isolated from differing sources.
  • the cardiac stem cells are endogenous stem cells or donor derived.
  • Cardiac stem cells are preferably indentified by at least one marker comprising connexin-43, N-cadherin, c-kit pos , CD3 neg , CD14 neg and CD68 neg .
  • the mesenchymal cells are autologous stem cells and are administered to a patient in a therapeutically effective dose to recruit endogenous stem cells to damaged tissue.
  • the mesenchymal stem cell and endogenous cardiac stem cells are optionally isolated and cultured ex-vivo and administered to a patient.
  • a method of treating damaged cardiac tissue comprises administering mesenchymal stem cells to cardiac tissue; stimulating cardiac stem cell in vivo proliferation; and, treating damaged cardiac tissue.
  • soluble factors from cultured mesenchymal stem cells are administered to cardiac tissue.
  • a mesenchymal stem cell factor stimulates the proliferation of cardiac stem cells in vivo.
  • an antibody or aptamer is specific, i.e. specifically binds a mesenchymal stem cell factor.
  • a mesenchymal stem cell factor stimulates the proliferation of cardiac stem cells in vitro.
  • a composition comprises mesenchymal stem cells and cardiac stem cells.
  • the composition comprises mesenchymal stem cells from one source, e.g. autologous, donor derived, etc;, and cardiac stem cells from another source, e.g. autologous, donor derived etc.
  • a mesenchymal stem cell comprises a polynucleotide encoding for a therapeutic agent, chemokine, growth factor or ligands thereof.
  • Figures IA- II are scans of photographs showing the trilineage differentiation of allogeneic MSCs.
  • Figure IA Cardiac lineage commitment of GFP pos ⁇ MSC expressing GATA- 4 (arrows) in the BZ of a treated heart 24h after transplantation.
  • Figure IB Cardiac committed ⁇ MSC undergoing symmetric division in the infarct zone of a treated heart 72h after transplantation.
  • Figure 1C GFP pos ⁇ MSC undergoing asymmetric division within the infarct zone of a 72h treated heart and giving rise to a cardiac committed (GATA-4 pos ) and an uncommitted daughter cell.
  • Figure ID Endothelial lineage commitment of ⁇ MSCs 72h after transplantation, indicated by the colocalization of GFP and KDR/factor VIII (arrows).
  • Figure IE Magnification of the lower right corner of Figure 1C.
  • Figure IF vascular smooth muscle cell commitment of ⁇ MSCs 72h after transplantation, as indicated by the co-localization of GFP with ⁇ -smooth muscle actin (arrows).
  • Figure IG Magnification of the lower right corner of Figure IE. Electrical coupling of aMSCs with surrounding cardiac tissue 24h after transplantation, as indicated by the co-localization of GFP and Connexin-43.
  • Figure II GFP pos cells in a regenerating zone (Rgz) 72h after transplantation are expressing ⁇ -sarcomeric actinin (arrows) and are mechanically coupled by N-cadherin with the infarcted (IZ) and border zone (BZ).
  • Figures 2A to 2J are scans of photographs showing the regeneration of cardiac muscle, vasculature and stem cell niches.
  • Figure 2A An immature GFP pos cardiac progenitor MSC expressing GATA-4 in the border zone of a treated heart 2 weeks after transplantation. Notice the adjoining c-kit pos CSC.
  • Figure 2B Immature MSCs in the border zone of a 2 week treated heart, expressing ⁇ -sarcomeric actinin (arrows).
  • Figure 2C Robust regeneration of the infarcted myocardium 2 weeks after transplantation, as demonstrated by the presence of Y-chromosome containing cardiac myocytes.
  • Figure 2D New coronary vessel formation 2 weeks after transplantation, as indicated by the Y-chromosome containing endothelial cell (arrow).
  • Figure 2E The chimeric myocardium consisted of mature cardiac myocytes, cardiac precursors and immature MSCs, all connected by connexin-43 with each other and the surrounding cardiac muscle (arrows).
  • Figure 2F Definite cardiac myocyte differentiation of MSCs 2 weeks after transplantation, as indicated by the existence of Y-chromosome containing mature cardiac myocytes.
  • Figure 2G Mobilization of c-kitpos cells at the sites of injury 2weeks after transplantation. C-kit cells were found in clusters in close proximity to GFP cells.
  • Figure 2H In contrast to the aMSCs treated animals, Placebo and control groups, showed significantly lower numbers of c-kit cells which were also isolated.
  • Figures 21, 2J The mobilized c-kit cells, often were connected with N-cadherin and connexin-43 with each other, the surrounding cardiac tissue and with GFP pos cells, indicating cardiac stem cell niches reconstitution.
  • Figures 3A to 3J show the long-term functional recovery following ⁇ MSCs transplantation.
  • Figures 3B, 3C are scans of photographs showing delayed contrast enhancement (DCE) MR images 3 months after experimental myocardial infarction (MI) ( Figure 3B) and same short axis view after 3 months of ⁇ MSCs therapy ( Figure 3C).
  • DCE delayed contrast enhancement
  • Figures 3E, 3F are scans of photographs showing representative 4mm-thick slices from the harvested hearts 12 weeks after MI, illustrating differences in LV diameter as well as in the extent of the scar size between the placebo and ⁇ MSCs-treated animals ( Figures 3E and 3F respectively).
  • Figure 3J is a scan of a photograph showing evidence of chimeric heart 12 weeks after transplantation, demonstrated by the presence of newly formed GATA-4 pos / ⁇ pos mature CM.
  • Figures 4A to 4E show the regeneration of large and small coronary vessels following ⁇ MSC transplantation.
  • Figures 4A-4C are scans of photographs showing confocal microscopy of large (Figure 4A), middle-sized ( Figure 4B) and capillary vessels (Figure 4C) in BZ of the ⁇ MSCs-treated group exhibiting Y pos cells into their structure. Large and middle-sized regenerated vessels co-localized with a smooth muscle actin and factor VIII.
  • Figures 4D, 4E are graphs showing quantification of new vessel densities per unit volume of myocardium. The majority of the regenerated vessels were detected in the BZ of the ⁇ MSCs treated hearts.
  • Figures 5 is a schematic illustration showing s summary of the in vivo results (panel insets a-f).
  • Figures 6A-6E show Laser Scanning Cytometry(LSC) for mapping cell trafficking.
  • Figures 6A-6C Within 72h post implantation, the GFP pos MSCs migrated from the endomyocardial site of injection to the infarcted subepicardial rim. No cells could be detected into the healthy surrounding myocardium indicating that MSCs trafficking was driven from damage signals.
  • Figures 6D, 6E Confocal Microscopy for anti- GFP and Y-chromosome was used to verify the detection of MSCs. Note that not all GFP pos cells are Y-chromosome pos and vice versa. The sensitivity of combined FISH/immunofluorescence detection was 45.5+2.1% of the total male cells.
  • Figures 7A to 7D are scans of photographs showing robust retention of ⁇ MSCs two weeks after intramyocardial transplantation.
  • Figure 7A shows hematoxylin and eosin histological stain demonstrating the tracing of an injection site.
  • Figure 7B shows the presence of GFP pos cells within the same injection confirming the origin of the exogenous cells.
  • Figures 7C, 7D Connexin-43 and ⁇ l-Integrin were used as markers of homing and engraftment of the MSCs in the host myocardium. Notice the strong presence of c-kit cells in Figure 7C.
  • Figures 8A to 8C show the fate of the allografts in the host myocardium during the first 72h after transplantation.
  • Figure 8A is a scan of a photograph showing immunofluorescence detection of the serine-10 phosphorylated Histone H3 (arrows) demonstrated the presence of mitotic ⁇ MSCs within an injection site, 72h after transplantation.
  • Figure 8B Co-localization of phospho-H3 and cleaved caspase-3 illustrated the premature chromatin condensation of an ⁇ MSC (arrow) and initiation of apoptosis 72h after transplantation. Arrowhead shows an adjacent ⁇ MSC in mitosis.
  • Figure 8C The number of apoptotic MSCs was counterbalanced by an analogous number of cells undergoing mitosis.
  • Figures 9A to 9D show the mobilization of endogenous c-kit pos CSCs 2 weeks after transplantation of MSCs.
  • Figures 9A-9C show the dramatic recruitment of c-kit pos cells at the sites of injection.
  • the c-kit cells were in clusters in the IZ ( Figure 9A) and BZ ( Figure 9B) of the treated hearts, but were mainly isolated in the healthy zones and/or the hearts of the nontreated animals (Figure 9C).
  • Figure 9D is a graph showing the distribution of the c-kit cells within the different zones of the treated and untreated animals.
  • Figures 1OA to 1OC show the stimulation of endogenous cardiac repair following ⁇ MSCs transplantation.
  • Figures 1OA, 1OB Besides the GFPpos MSCs, the regenerated CSC niches were containing clusters of c-kit pos cells which were co-localized with MDRl pos and GATA-4 pos .
  • Figure 1OC Quantification of the c-kit pos / GATA-4 pos cardiac precursor cells between groups, demonstrated an active endogenous repair process in the MSCs treated animals which was commenced, but not restricted, in the BZ of the infarcted hearts.
  • Figures HA to HC show the long-term retention of ⁇ MSCs.
  • Figure 1 IB Distribution of the ⁇ MSCs in the chronic infarcted myocardium. Cells were detected in the IZ and BZ but not the remote healthy zones.
  • Figure 11C Newly formed cardiomyocytes were fully functional and integrated into the host myocardium; Demonstration of electrical coupling of ⁇ MSCs with the resident cardiomyocytes via the development of connexin-43 gap junctions (arrows).
  • “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • heart disease refers to any type of heart disease including cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, diseases resulting in remodeling of the heart, etc.
  • Diseases of the heart can be due to any reason, such as for example, damage to cardiac tissue such as a loss of contractility (e.g., as might be demonstrated by a decreased ejection fraction).
  • Cardiac damage or disorder characterized by insufficient cardiac function includes any impairment or absence of a normal cardiac function or presence of an abnormal cardiac function.
  • Abnormal cardiac function can be the result of disease, injury, and/or aging.
  • abnormal cardiac function includes morphological and/or functional abnormality of a cardiomyocyte, a population of cardiomyocytes, or the heart itself.
  • Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of cardiomyocytes, abnormal growth patterns of cardiomyocytes, abnormalities in the physical connection between cardiomyocytes, under- or over-production of a substance or substances by cardiomyocytes, failure of cardiomyocytes to produce a substance or substances which they normally produce, and transmission of electrical impulses in abnormal patterns or at abnormal times.
  • Abnormalities at a more gross level include dyskinesis, reduced ejection fraction, changes as observed by echocardiography (e.g., dilatation), changes in EKG, changes in exercise tolerance, reduced capillary perfusion, and changes as observed by angiography.
  • ischemic heart disease e.g., angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertensive heart disease, pulmonary heart disease (cor pulmonale), valvular heart disease, e.g., rheumatic fever, mitral valve prolapse, calcification of mitral annulus, carcinoid heart disease, infective endocarditis, congenital heart disease, myocardial disease, e.g., myocarditis, dilated cardiomyopathy, hypertensive cardiomyopathy, cardiac disorders which result in congestive heart failure, and tumors of the heart, e.g., primary sarcomas and secondary tumors.
  • Heart damage also includes wounds, such as for example, knife wound; biological (e.g. viral; autoimmune diseases) or chemical (e.g. chemotherapy, drugs); surgery; transplantation and the like.
  • Myocardial ischemia refers to a lack of oxygen flow to the heart which results in myocardial ischemic damage.
  • myocardial ischemic damage includes damage caused by reduced blood flow to the myocardium.
  • causes of myocardial ischemia and myocardial ischemic damage include: decreased aortic diastolic pressure, increased intraventricular pressure and myocardial contraction, coronary artery stenosis (e.g., coronary ligation, fixed coronary stenosis, acute plaque change (e.g., rupture, hemorrhage), coronary artery thrombosis, vasoconstriction), aortic valve stenosis and regurgitation, and increased right atrial pressure.
  • Non-limiting examples of adverse effects of myocardial ischemia and myocardial ischemic damage include: myocyte damage (e.g., myocyte cell loss, myocyte hypertrophy, myocyte cellular hyperplasia), angina (e.g., stable angina, variant angina, unstable angina, sudden cardiac death), myocardial infarction, and congestive heart failure. Damage due to myocardial ischemia may be acute or chronic, and consequences may include scar formation, cardiac remodeling, cardiac hypertrophy, wall thinning, dilatation, and associated functional changes.
  • the existence and etiology of acute or chronic myocardial damage and/or myocardial ischemia may be diagnosed using any of a variety of methods and techniques well known in the art including, e.g., non-invasive imaging (e.g., MRI, echocardiography), angiography, stress testing, assays for cardiac- specific proteins such as cardiac troponin, and clinical symptoms. These methods and techniques as well as other appropriate techniques may be used to determine which subjects are suitable candidates for the treatment methods described herein.
  • “Stem cell niche” refers to the microenvironment in which stem cells are found, which interacts with stem cells to regulate stem cell fate. (See, for example, Kendall Powell, Nature 435, 268 - 270 (2005).
  • the word 'niche' can be in reference to the in vivo or in vitro stem cell microenvironment.
  • various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus.
  • stem cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding microenvironment actively signals to stem cells to either promote self renewal or differentiation to form new tissues.
  • stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood, specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair.
  • the niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation.
  • stem-cell therapies It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapies.
  • Bio samples include solid and body fluid samples.
  • the biological samples used in the present invention can include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • solid biological samples include, but are not limited to, samples taken from tissues of the central nervous system, bone, breast, kidney, cervix, endometrium, head/neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose, lymph node, uterus, ovary, adrenal gland, testes, tonsils and thymus.
  • body fluid samples include, but are not limited to blood, serum, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears.
  • BMDC bone marrow derived progenitor cell
  • bone marrow derived stem cell refers to a primitive stem cell with the machinery for self-renewal constitutively active. Included in this definition are stem cells that are totipotent, pluripotent and precursors.
  • a "precursor cell” can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell.
  • the term “precursor cell population” refers to a group of cells capable of developing into a more mature cell.
  • a precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e.
  • the term “totipotent cell” refers to a cell capable of developing into all lineages of cells.
  • the term “totipotent population of cells” refers to a composition of cells capable of developing into all lineages of cells.
  • the term “pluripotent cell” refers to a cell capable of developing into a variety (albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and thrombocytic lineages).
  • Bone marrow derived stem cells contain two well- characterized types of stem cells.
  • Mesenchymal stem cells normally form chondrocytes and osteoblasts.
  • Hematopoietic stem cells are of mesodermal origin that normally give rise to cells of the blood and immune system (e.g., erythroid, granulocyte/macrophage, magakaryocite and lymphoid lineages).
  • hematopoietic stem cells also have been shown to have the potential to differentiate into the cells of the liver (including hepatocytes, bile duct cells), lung, kidney (e.g., renal tubular epithelial cells and renal parenchyma), gastrointestinal tract, skeletal muscle fibers, astrocytes of the CNS, Purkinje neurons, cardiac muscle (e.g., cardiomyocytes), endothelium and skin.
  • autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
  • xenogeneic cell refers to a cell that derives from a different animal species than the animal species that becomes the recipient animal host in a transplantation or vaccination procedure.
  • allogeneic cell refers to a cell that is of the same animal species but genetically different in one or more genetic loci as the animal that becomes the "recipient host”. This usually applies to cells transplanted from one animal to another non-identical animal of the same species.
  • genotypic cell refers to a cell which is of the same animal species and has the same genetic composition for most genotypic and phenotypic markers as the animal who becomes the recipient host of that cell line in a transplantation or vaccination procedure. This usually applies to cells transplanted from identical twins or may be applied to cells transplanted between highly inbred animals.
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” The
  • diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • ameliorated refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a normalized value for example a value obtained in a healthy patient or individual
  • telomere binding means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A,” the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.
  • an antibody that "specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • Embodiments of the invention provide for compositions comprising mesenchymal stem cells (MSCs) utilized, in some aspects, for the acceleration of the preparation of cardiac stem cells (CSCs.) Enhancing the CSCs in vitro will improve their utility.
  • MSCs can also be used to proliferate CSCs in vivo.
  • the CSCs proliferate and differentiate into cardiac cells, e.g. cardiomyocytes.
  • the results described herein show that injections of MSCs into pig hearts caused massive proliferation of CSCs.
  • MSCs bind to and form complexes with CSCs, and cause their proliferation and differentiation into cardiac myocytes.
  • the cardiac stem cell proliferation can be due to a mesenchymal stem cell factor, a receptor ligand interaction between the MSCs and CSCs or other cells in the cardiac tissue, or combinations of both.
  • GFP labeled male allogeneic adult bone marrow derived mesenchymal stem cells were injected into female pigs following myocardial infarction (MI) ( Figure 5). It was hypothesized that allogeneic adult bone marrow derived mesenchymal stem cells ( ⁇ MSCs) were true adult precursor cells which could differentiate into three cardiac cell lineages, and that ⁇ MSCs participate in cardiac recovery by forming cell-cell interactions with existing myocardial elements including with endogenous precursor cells.
  • a stem cell is a cell from the embryo, fetus, or adult that has, under certain conditions, the ability to reproduce itself for long periods or, in the case of adult stem cells, throughout the life of the organism. It also can give rise to specialized cells that make up the tissues and organs of the body.
  • a pluripotent stem cell has the ability to give rise to types of cells that develop from the three germ layers (mesoderm, endoderm, and ectoderm) from which all the cells of the body arise.
  • the only known sources of human pluripotent stem cells are those isolated and cultured from early human embryos and from fetal tissue that was destined to be part of the gonads.
  • An embryonic stem cell is derived from a group of cells called the inner cell mass, which is part of the early (4- to 5-day) embryo called the blastocyst. Once removed from the blastocyst the cells of the inner cell mass can be cultured into embryonic stem cells. These embryonic stem cells are not themselves embryos.
  • An adult stem cell is an undifferentiated (unspecialized) cell that occurs in a differentiated (specialized) tissue, renews itself, and becomes specialized to yield all of the specialized cell types of the tissue in which it is placed when transferred to the appropriate tissue.
  • Adult stem cells are capable of making identical copies of themselves for the lifetime of the organism. This property is referred to as "self -renewal.”
  • Adult stem cells usually divide to generate progenitor or precursor cells, which then differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions, e.g., muscle cell contraction or nerve cell signaling.
  • Sources of adult stem cells include bone marrow, blood, the cornea and the retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, the lining of the gastrointestinal tract and pancreas.
  • Stem cells from the bone marrow are the most-studied type of adult stem cells. They can be used clinically to restore various blood and immune components to the bone marrow via transplantation.
  • HSC hematopoietic stem cells
  • MSC stromal (mesenchymal) stem cells
  • both types of marrow-derived stem cells recently have demonstrated extensive plasticity and multipotency in their ability to form the same tissues.
  • the marrow, located in the medullary cavity of bones, is the sole site of hematopoiesis in adult humans.
  • Hematopoietically active (red) marrow regresses after birth until late adolescence after which time it is focused in the lower skull vertebrae, shoulder and pelvic girdles, ribs, and sternum.
  • Fat cells replace hematopoietic cells in the bones of the hands, feet, legs and arms (yellow marrow). Fat comes to occupy about fifty percent of the space of red marrow in the adult and further fatty metamorphosis continues slowly with aging. In very old individuals, a gelatinous transformation of fat to a mucoid material may occur (white marrow).
  • Yellow marrow can revert to hematopoietically active marrow if prolonged demand is present such as with hemolytic anemia.
  • hematopoiesis can be expanded by increasing the volume of red marrow and decreasing the development (transit) time from progenitor to mature cell.
  • the marrow stroma consists principally of a network of sinuses that originate at the endosteum from cortical capillaries and terminate in collecting vessels that enter the systemic venous circulation.
  • the trilaminar sinus wall is composed of endothelial cells; an underdeveloped, thin basement membrane, and adventitial reticular cells that are fibroblasts capable of transforming into adipocytes.
  • the endothelium and reticular cells are sources of hematopoietic cytokines. Hematopoiesis takes place in the intersinus spaces and is controlled by a complex array of stimulatory and inhibitory cytokines, cell-to-cell contacts and the effects of extracellular matrix components on proximate cells.
  • lymphohematopoietic stem cells differentiate into all of the blood cell types. Mature cells are produced and released to maintain steady state blood cell levels. The system may meet increased demands for additional cells as a result of blood loss, hemolysis, inflammation, immune cytopenias, and other causes.
  • a "progenitor or precursor” cell occurs in fetal or adult tissues and is partially specialized; it divides and gives rise to differentiated cells.
  • a progenitor/precursor cell divides, it can form more progenitor/precursor cells or it can form two specialized cells.
  • Progenitor/precursor cells can replace cells that are damaged or dead, thus maintaining the integrity and functions of a tissue such as liver or brain.
  • Mesenchymal stem cells are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into any of the specific types of mesenchymal or connective tissues (i.e. the tissues of the body that support the specialized elements; particularly adipose, osseous, cartilaginous, elastic, and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines
  • mesenchymal stem cells are isolated from bone marrow of adult patients.
  • the cells are passed through a density gradient to eliminate undesired cell types.
  • the cells are preferably, plated and cultured in appropriate media.
  • the cells are cultured for at least one day, preferably, about three to about seven days, and removing non-adherent cells. The adherent cells are plated and expanded.
  • Umbilical cord blood is an abundant source of hematopoietic stem cells.
  • the stem cells obtained from umbilical cord blood and those obtained from bone marrow or peripheral blood appear to be very similar for transplantation use.
  • Placenta is an excellent readily available source for mesenchymal stem cells.
  • mesenchymal stem cells can be derivable from adipose tissue and bone marrow stromal cells and speculated to be present in other tissues. While there are dramatic qualitative and quantitative differences in the organs from which adult stem cells can be derived, the initial differences between the cells may be relatively superficial and balanced by the similar range of plasticity they exhibit.
  • Homogeneous human mesenchymal stem cell compositions which serve as the progenitors for all mesenchymal cell lineages.
  • MSCs are identified by specific cell surface markers which are identified with unique monoclonal antibodies.
  • the homogeneous MSC compositions are obtained by positive selection of adherent marrow or periosteal cells which are free of markers associated with either hematopoietic cell or differentiated mesenchymal cells.
  • These isolated mesenchymal cell populations display epitopic characteristics associated with only mesenchymal stem cells, have the ability to regenerate in culture without differentiating, and have the ability to differentiate into specific mesenchymal lineages when either induced in vitro or placed in vivo at the site of damaged tissue.
  • pluripotent mesenchymal stem cells are separated from other cells in the bone marrow or other MSC source.
  • Bone marrow cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary spaces .
  • Other sources of human mesenchymal stem cells include embryonic yolk sac, placenta, umbilical cord, fetal and adolescent skin, and blood.
  • the mesenchymal stem cells can be isolated and purified by different methods. A preferred method has been described in detail in the examples section which follows. Other methods include, providing a tissue specimen containing mesenchymal stem cells, adding cells from the tissue specimen to a medium which contains factors that stimulate mesenchymal stem cell growth without differentiation and allows, when cultured, for the selective adherence of only the mesenchymal stem cells to a substrate surface, culturing the specimen-medium mixture, and removing the non-adherent matter from the substrate surface.
  • the mesenchymal stem cells are derived from one or sources comprising: autologous, heterologous, syngeneic, allogeneic or xenogeneic sources. These sources can include cell lines. As used herein, "source” refers to the animal in which these stem cells were obtained from, including human.
  • Differentiation of mesenchymal stem cells to the cardiac lineage is controlled by factors present in the cardiac environment. Local chemical, electrical and mechanical environmental influences alter pluripotent MSCs and convert the cells administered to the heart into the cardiac lineage.
  • mesenchymal stem cells differentiate into lineages of cells that make up the different heart tissues.
  • the lineages are identified by cardiac cell specific markers comprising cardiac transcription factor GATA-4, MDRl; endothelial cell markers Factor VIII and KDR; vascular smooth muscle marker ⁇ -smooth muscle actin; or cardiomyocyte marker ⁇ -sarcomeric actinin.
  • cardiac cell specific markers comprising cardiac transcription factor GATA-4, MDRl; endothelial cell markers Factor VIII and KDR; vascular smooth muscle marker ⁇ -smooth muscle actin; or cardiomyocyte marker ⁇ -sarcomeric actinin.
  • detection of expression of cardiomyocyte specific proteins is achieved using antibodies to, for example, myosin heavy chain monoclonal antibody MF 20 (MF20), sarcoplasmic reticulum calcium ATPase (SERCAl) (mnAb 10Dl) or gap junctions using antibodies to connexin 43.
  • MF20 myosin heavy chain monoclonal antibody MF 20
  • markers for cardiomyocytes comprise cardiac troponin I (cTnl), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, ⁇ l -adrenoceptor ⁇ l-AR), ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF).
  • cardiac troponin I cTnl
  • cTnT cardiac troponin T
  • MHC sarcomeric myosin heavy chain
  • GATA-4 GATA-4
  • Nkx2.5 N-cadherin
  • ⁇ l -adrenoceptor ⁇ l-AR ⁇ l -adrenoceptor ⁇ l-AR
  • ANF the MEF-2 family of transcription factors
  • CK-MB creatine kinase MB
  • myoglobin myoglobin
  • GATA transcription factor includes members of the GATA family of zinc finger transcription factors. GATA transcription factors are involved in the development of several mesodermally derived cell lineages. Preferably, GATA transcription factors include GATA-4 and/or GATA-6. The GATA-6 and GATA-4 proteins share high-level amino acid sequence identity over a proline-rich region at the amino terminus of the protein that is not conserved in other GATA family members.
  • soluble factors from mesenchymal stem cell cultures are administered to a patient with heart disease or disorders thereof. The factors are administered in an effective amount resulting in the localization, proliferation and maturation of cardiac stem cells into cells of the damaged heart tissue.
  • the mesenchymal cells are administered to a patient, for treating heart disease or disorders thereof.
  • the cells can be autologous or donor derived.
  • the administration of the mesenchymal stem cells results in the recruitment of cardiac stem cells to the damaged tissues, accelerated proliferation and differentiation of the cardiac stem cells and the repair of the damaged tissues.
  • the cardiac stem cells are endogenous stem cells.
  • the cardiac stem cells can be donor derived.
  • the combinations of origins of the mesenchymal stem cells and cardiac stem cells can be in any combinations.
  • mesenchymal stem cells are autologous and the cardiac stem cells are endogenous.
  • the mesenchymal stem cells are donor derived and the cardiac stem cells are endogenous.
  • the mesenchymal stem cells are autologous and the cardiac stem cells are donor derived.
  • the mesenchymal stem cells comprise both autologous and donor derived cells and the cardiac stem cells comprise donor derived and endogenous cells.
  • soluble factors from mesenchymal stem cells are administered to the damaged heart tissue for recruiting, accelerating proliferation and differentiation of cardiac stem cells.
  • cardiac stem cells are identified by markers comprising: c- kit pos , CD3 neg , CD14 neg and CD68 neg .
  • Cardiac injury promotes tissue responses which enhance myogenesis using implanted MSCs.
  • MSCs are introduced to the infarct zone to reduce the degree of scar formation and to augment ventricular function. New muscle is thereby created within an infarcted myocardial segment.
  • MSCs are directly infiltrated into the zone of infarcted tissue. The integration and subsequent differentiation of these cells is characterized, as described above. Timing of intervention is designed to mimic the clinical setting where patients with acute myocardial infarction would first come to medical attention, receive first-line therapy, followed by stabilization, and then intervention with myocardial replacement therapy if necessary.
  • the left ventricle is primarily responsible for pumping blood under pressure through the body's circulatory system. It has the thickest myocardial walls and is the most frequent site of myocardial injury resulting from congestive heart failure.
  • the degree of advance or severity of the congestive heart failure ranges from those cases where heart transplantation is indicated as soon as a suitable donor organ becomes available to those where little or no permanent injury is observed and treatment is primarily prophylactic.
  • the severity of resulting myocardial infarction, i.e. the percentage of muscle mass of the left ventricle that is involved can range from about 1 to about 80 percent.
  • the severity of the infarction is significantly affected by which vessel(s) is involved and how much time has passed before treatment intervention is begun.
  • the mesenchymal stem cells used in accordance with the invention are, in order of preference, autologous, allogeneic or xenogeneic, and the choice can largely depend on the urgency of the need for treatment.
  • a patient presenting an imminently life threatening condition may be maintained on a heart/lung machine while sufficient numbers of autologous MSCs are cultured or initial treatment can be provided using other than autologous MSCs.
  • the MSC therapy of the invention can be provided by several routes of administration, including the following.
  • intracardiac muscle injection which avoids the need for an open surgical procedure, can be used where the MSCs are in an injectible liquid suspension preparation or where they are in a biocompatible medium which is injectible in liquid form and becomes semi- solid at the site of damaged myocardium.
  • a conventional intracardiac syringe or a controllable arthroscopic delivery device can be used so long as the needle lumen or bore is of sufficient diameter (e.g. 30 gauge or larger) that shear forces will not damage the MSCs.
  • the injectible liquid suspension MSC preparations can also be administered intravenously, either by continuous drip or as a bolus.
  • MSC delivery preparations are available options.
  • a dose range is a volume of about 20 to about 50 ⁇ l of injectible suspension containing about 10-40 xlO 6 MSCs/ml.
  • concentration of cells per unit volume, whether the carrier medium is liquid or solid remains within substantially the same range.
  • the amount of MSCs delivered will usually be greater when a solid, "patch" type application is made during an open procedure, but follow-up therapy by injection will be as described above.
  • the frequency and duration of therapy will, however, vary depending on the degree (percentage) of tissue involvement, as already described (e.g. 5-40% left ventricular mass).
  • the injection medium can be any pharmaceutically acceptable isotonic liquid. Examples include phosphate buffered saline (PBS), culture media such as DMEM (preferably serum-free), physiological saline or 5% dextrose in water.
  • PBS phosphate buffered saline
  • DMEM preferably serum-free
  • physiological saline preferably 5% dextrose in water.
  • the isolated and culture expanded mesenchymal stem cells can be utilized for the implantation of various prosthetic devices. For example, using porous ceramic structures filled with culture-expanded human mesenchymal stem cells, and implanting these structures in areas where there is extensive tissue damage.
  • stem cells can be used with the mesenchymal stem cells in the treatment of heart diseases and disorders thereof.
  • the stem cells are totipotent or pluripotent stem cells.
  • Stem cells are undifferentiated immature cells, capable of self renewal (division without limit) and differentiation
  • stem cells are either monopotent, bipotent or pluripotent.
  • Monopotent and bipotent stem cells are more restricted in development and give rise to one or two types of specialized cells, respectively.
  • the pluripotent stem cells can differentiate into many different types of cells, giving rise to tissue (which constitute organs) or in the case of totipotent stem cells, the whole organism.
  • Pluripotent stem cells unlike monopotent or bipotent, are capable of multilineage differentiation, giving rise to a tissue which would consist of a collection of cells of different types or lineages.
  • a stem cell such as a pluripotent stem cell
  • a pluripotent stem cell has the following four characteristics: (i) it is an undifferentiated cell— i.e. it is not terminally differentiated; (ii) it has the ability to divide without limit; (iii) it has the ability to give rise to differentiated progeny; and (iv) when it divides each daughter has a choice: it can either remain as stem cell like its parent or it can embark on a course leading to differentiation.
  • the hematopoietic stem cell is an example of a pluripotent stem cell which is found among marrow cells and gives rise to all the various blood cells (including leucocytes and erythrocytes).
  • Hemopoietic stem cells can be extracted by isolation from (i) bone marrow, (ii) growth factor mobilized peripheral blood or (iii) cord blood (placenta).
  • hemopoietic stem cells have been prepared from Embryonic Stem cells (ES), which are extracted from embryos obtained using in vitro fertilization techniques. These undifferentiated cells are capable of multi-lineage differentiation and reconstitution of all body tissue i.e. are totipotent.
  • LSCs lymphoid stem cells
  • LSCs myeloid stem cells
  • T cells T cells
  • B cells eosinophils
  • basophils neutrophils
  • megakaryocytes monocytes
  • granulocytes mast cells
  • lymphocytes granulocytes
  • stem cells include neural stem cells, multipotent stem cells that can generate neurons, atrocytes and oligodendrocytes (Nakafuku and Nakamura, 1995, J. Neurosci Res., vol 41(2): 153-68; Anderson, 1994, FASEB J., vol 8(10): 707-13; Morshead et al, 1994, Neuron, VoI 13(5): 1071-82).
  • Skeletal muscle satellite cells are another type of stem cell, more specifically a distinct class of myogenic cells that are maintained as quiescent stem cells in the adult and can give rise to new muscle cells when needed (Bischoff, 1986, Dev Biol., vol 115(1): 129-39).
  • Other types of stem cells are epithelial stem cells, a subset of basal cells, and mesenchymal stem cells.
  • Embryonic stem (ES) cells are routinely used in the production of transgenic animals. ES cells have been shown to differentiate in vitro into several cell types including lymphoid precursors (Potocnik et al, 1994, EMBO J., vol 13(22): 5274-83) and neural cells.
  • stage-specific markers such as stage-specific embryonic markers 3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoproteins TRA-1-60 and TRA-1-81 and alkaline phosphatase (Andrews et al, 1984, Hybridoma, vol 3: 347-361; Kannagi et al., 1983, EMBO J., vol 2: 2355-2361; Fox et al., 1984, Dev. Biol., vol 103: 263-266; Ozawa et al., 1985, Cell. Differ., vol 16: 169-173).
  • stage-specific embryonic markers 3 and 4 SSEA-3 and SSEA-4
  • TRA-1-60 and TRA-1-81 and alkaline phosphatase
  • association here means the cells expressing or capable of expressing, or presenting or capable of being induced to present, or comprising, the respective antigen(s).
  • Most undifferentiated cells and differentiated cells comprise Major Histocompatibility Complex (MHC) Class I antigens and/or Class II antigens. If these antigens are associated with those cells then they are called Class I + and/or Class H + cells.
  • MHC Major Histocompatibility Complex
  • Each specific antigen associated with an undifferentiated cell or a differentiated cell can act as a marker.
  • different types of cells can be distinguished from each other on the basis of their associated particular antigen(s) or on the basis of a particular combination of associated antigens. Examples of these marker antigens include the antigens CD34, CD 19 and CD3.
  • CD34 + , CD19 + and CD3 + cells are called CD34 + , CD19 + and CD3 + cells respectively. If these antigens are not present then these cells are called CD34 " , CD 19 " and CD3 " cells respectively.
  • Some of the markers identified on myeloid stem cells comprise CD34 + DR + , CD13 + , CD33 + , CD7 + and TdT + cells.
  • PSCs are CD34 + DR " TdT " cells (other useful markers being CD38 " and CD36 + ).
  • LSCs are DR + , CD34 + and TdT + cells (also CD38 + ).
  • Embryonic stem cells express SSEA-3 and SSEA-4, high molecular weight glycoproteins TRA-1-60 and TRA-1-81 and alkaline phosphatase. They also do not express SSEA-I, the presence of which is an indicator of differentiation. Other markers are known for other types of stem cells, such as Nestein for neuroepithelial stem cells (J. Neurosci, 1985, VoI 5: 3310). Mesenchymal stem cells are also positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a and CD124, for example, and negative for CD34, CD45 and CD14. Stem cells may further be isolated for transduction and differentiation using known methods.
  • bone marrow cells are isolated by sacrificing the mouse and cutting the leg bones with a pair of scissors.
  • Stem cells may also be isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4 + and CD8+ (T cells), CD45 + (panB cells), GR-I (granulocytes), and lad (differentiated antigen presenting cells).
  • T cells CD4 + and CD8+
  • CD45 + panB cells
  • GR-I granulocytes
  • lad differentiated antigen presenting cells
  • CD34 + hematopoietic stem cells can be obtained from a variety of sources including cord blood, bone marrow, and mobilized peripheral blood. Purification of CD34 + cells can be accomplished by antibody affinity procedures. An affinity column isolation procedure for isolating CD34 + cells is described by Ho et al, Stem Cells 13 (suppl. 3): 100-105(1995). See also, Brenner, Journal of Hematotherapy 2: 7-17 (1993). Methods for isolating, purifying and culturally expanding mesenchymal stem cells are known.
  • many cells can be identified by morphological characteristics.
  • the identification of cells using microscopy, optionally with staining techniques is an extremely well developed branch of science termed histology and the relevant skills are widely possessed in the art.
  • Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation. If desired, a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude" separation. For example, magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed.
  • Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • cardiotropic factors can be included in the culture medium if it is desired by a user to differentiate the stem cells. These are factors that either alone or in combination enhance proliferation or survival of cardiomyocyte type cells, or inhibit the growth of other cell types. The effect may be due to a direct effect on the cell itself, or due to an effect on another cell type, which in turn enhances cardiomyocyte formation. For example, factors that induce the formation of hypoblast or epiblast equivalent cells, or cause these cells to produce their own cardiac promoting elements, all come within the rubric of cardiotropic factors.
  • TGF- ⁇ ligands that affect DNA methylation and altering expression of cardiomyocyte -related genes TGF- ⁇ ligands (exemplified by TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3 and other members of the TGF- ⁇ superfamily). Ligands bind a TGF- ⁇ receptor activate Type I and Type II serine kinases and cause phosphorylation of the Smad effector.
  • Morphogens like Activin A and Activin B members of the TGF- ⁇ superfamily
  • Insulin-like growth factors such as IGF II
  • Bone morphogenic proteins members of the TGF- ⁇ superfamily, exemplified by BMP-2 and BMP-4
  • Fibroblast growth factors exemplified by bFGF, FGF-4, and FGF-8
  • MAPK mitogen- activated proteins kinase
  • Platelet- derived growth factor exemplified by PDGF ⁇
  • Natriuretic factors exemplified by atrial natriuretic factor (ANF), brain natriuretic peptide (BNP).
  • the cells can be cocultured with cells (such as endothelial cells of various kinds) that secrete factors enhancing cardiomyocyte differentiation.
  • Nucleotide analogs that affect DNA methylation (and thereby influence gene expression) can effectively be used to increase the proportion of cardiomyocyte lineage cells that emerge following initial differentiation. For example, it has been found that inclusion of 5-aza-deoxy-cytidine in the culture medium increases the frequency of contracting cells in the population, and expression of cardiac ⁇ MHC.
  • cardiotropic agents include use of a morphogen like Activin A and a plurality of growth factors, such as those included in the TGF- ⁇ and IGF families during the early commitment stage, optionally supplemented with additional cardiotropins such as one or more fibroblast growth factors, bone morphogenic proteins, and platelet-derived growth factors.
  • a morphogen like Activin A and a plurality of growth factors such as those included in the TGF- ⁇ and IGF families during the early commitment stage, optionally supplemented with additional cardiotropins such as one or more fibroblast growth factors, bone morphogenic proteins, and platelet-derived growth factors.
  • IGF II insulin-like growth factor II
  • IGF II insulin-like growth factor II
  • Wnt proteins normally stabilize and cause nuclear translocation of a cytoplasmic molecule, ⁇ -catenin, which comprises a portion of the transcription factor TCF. This changes transcriptional activity of multiple genes.
  • ⁇ -catenin is phosphorylated by the kinase GSK3 ⁇ , which both destabilizes ⁇ - catenin and keeps it in the cytoplasm.
  • Wnt activators like IL-2 apparently limit cardiomyocyte differentiation, it is believed that culturing with Wnt antagonists can increase the extent or proportion of cardiomyocyte differentiation of hES cells.
  • Wnt signaling can be inhibited by injection of synthetic mRNA encoding either DKK-I or Crescent (secreted proteins that bind and inactivate Wnts) (Schneider et al., Genes Dev. 15:304, 2001), or by infection with a retrovirus encoding DKK-I (Marvin et al., Genes Dev. 15:316, 2001).
  • the Wnt pathway can be inhibited by increasing the activity of the kinase GSK3 ⁇ , for example, by culturing the cells with factors such as IL-6 or glucocorticoids.
  • factors such as IL-6 or glucocorticoids.
  • the combinations and amounts of such compounds that are effective for enriching cardiomyocyte production can be determined empirically by culturing undifferentiated or early differentiated embryonic stem cells or their progeny in a culture environment incorporating such factors, and then determining whether the compound has increased the number of cardiomyocyte lineage cells in the population according to the phenotypic markers listed below.
  • cardiomyocyte lineage cells Following initial differentiation (and before or after a separation step, if employed), it is possible to increase the percentage of cardiomyocyte lineage cells by culturing in an environment containing a "cardiomyocyte enrichment agent". This is a factor in the medium or on a surface substrate that promotes the outgrowth of the desired cell type— either by facilitating proliferation of cardiomyocyte lineage cells, or by inhibiting the growth (or causing apoptosis) of cells of other tissue types.
  • cardiotropic factors listed above are suitable for this purpose.
  • certain compounds known beneficial to cardiomyocytes in vivo, or their analogs Included are compounds capable of forming a high energy phosphate bond (such as creatine); an acyl group carrier molecule (such as carnitine); and a cardiomyocyte calcium channel modulator (such as taurine).
  • Cardiomyocyte specific markers comprise: Cardiac troponin I (cTnl), a subunit of troponin complex that provides a calcium- sensitive molecular switch for the regulation of striated muscle contraction.
  • Cardiac troponin T (cTnT) Atrial natriuretic factor (ANF), a hormone expressed in developing heart and fetal cardiomyocytes but down-regulated in adults. It is considered a good marker for cardiomyocytes because it is expressed in a highly specific manner in cardiac cells but not skeletal myocytes.
  • the cells will also typically express at least one (and often at least 3, 5, or more) of the following markers: sarcomeric myosin heavy chain (MHC) Titin, tropomyosin, ⁇ -actinin, and desmin, GATA-4, a transcription factor that is highly expressed in cardiac mesoderm and persists in the developing heart. It regulates many cardiac genes and plays a role in cardiogenesis Nkx2.5, a cardiac transcription factor expressed in cardiac mesoderm during early mouse embryonic development, which persists in the developing heart.
  • MHC myosin heavy chain
  • MEF-2A, MEF- 2B, MEF-2C, MEF-2D transcription factors that are expressed in cardiac mesoderm and persist in developing heart N-cadherin, which mediates adhesion among cardiac cells Connexin 43, which forms the gap junction between cardiomyocytes.
  • ⁇ l -adrenoceptor ( ⁇ l- AR) creatine kinase MB (CK-MB) and myoglobin, which are elevated in serum following myocardial infarction.
  • Other markers that may be positive on cardiomyocytes and their precursors include ⁇ -cardiac actin, early growth response-I, and cyclin D2.
  • Tissue-specific markers can be detected using any suitable immunological technique- such as flow immunocytochemistry or affinity adsorption for cell- surface markers, immunocytochemistry (for example, of fixed cells or tissue sections) for intracellular or cell- surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • Immunlogical technique such as flow immunocytochemistry or affinity adsorption for cell- surface markers, immunocytochemistry (for example, of fixed cells or tissue sections) for intracellular or cell- surface markers, Western blot analysis of cellular extracts, and enzyme-linked immunoassay, for cellular extracts or products secreted into the medium.
  • Expression of an antigen by a cell is said to be antibody-detectable if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, and optionally using a labeled secondary antibody or other conjugate (such as a biotin
  • tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence- specific primers in standard amplification methods. See U.S. Pat. No. 5,843,780 for details of general technique. Sequence data for other markers listed in this disclosure can be obtained from public databases such as GenBank (URL www.ncbi.nlm.nih. gov:80/entrez). Expression at the mRNA level is said to be detectable according to one of the assays described in this disclosure if the performance of the assay on cell samples according to standard procedures in a typical controlled experiment results in clearly discernable hybridization or amplification product.
  • GenBank URL www.ncbi.nlm.nih. gov:80/entrez
  • tissue-specific markers as detected at the protein or mRNA level is considered positive if the level is at least 2-fold, and preferably more than 10- or 50-fold above that of a control cell, such as an undifferentiated pluripotent stem celll or other unrelated cell type.
  • markers Once markers have been identified on the surface of cells of the desired phenotype, they can be used for immuno selection to further enrich the population by techniques such as immunopanning or antibody-medicated fluorescence-activated cell sorting.
  • the mesenchymal stem cells comprise therapeutic genes for delivery into the body, such as for example, the heart.
  • the therapeutic gene is a transgene.
  • the delivery cells e.g. the mesenchymal stem cells are removed from the body, and a therapeutic transgene is introduced into them via vehicles well known to those skilled in the art such as those used in direct-gene-transfer methods.
  • the genetically modified cells are tested and then allowed to grow and multiply and, finally, are infused back into the patient.
  • allogeneic cells that bear normal, endogenous genes can reverse a deficiency in a particular target tissue. Use of cells bearing either transgenes or normal, endogenous genes is referred to herein as gene therapy.
  • Gene therapy using genetically modified cells offers several unique advantages over direct gene transfer into the body.
  • the mesenchymal stem cells express stem cell recruiting factors, growth factors, therapeutic factors, endogenous factors such as, for example, cardiac troponin I (cTnl), cardiac troponin T (cTnT), atrial natriuretic factor (ANF), and the like.
  • endogenous factors such as, for example, cardiac troponin I (cTnl), cardiac troponin T (cTnT), atrial natriuretic factor (ANF), and the like.
  • the methods according to the present invention are useful for targeting a gene of interest (either a transgene or an endogenous gene) to a tissue in a mammal by introducing a cell comprising the gene of interest to the mammal.
  • Such methods are useful for treating a disease characterized by a deficiency in a gene product in a mammal by administering a cell comprising a functional gene encoding the gene product into the mammal and administering a glycoconjugate to the mammal.
  • Stem cells may be used as a vehicle for delivering genes to specific tissues in the body.
  • Stem cell-based therapies are a major area of investigation in cancer research.
  • Embodiments of the invention further provide localizing of transfused cells such as stem cells to provide a functional gene to a patient suffering from a disease caused by a lack of that gene, or, recruitment of endogenous stem cells to the damaged heart tissue.
  • transfused cells such as stem cells
  • the present invention also provides the ability to direct the localization of the transfused cells such as allogeneic stem cells that have a stable, normal gene. Such transfused cells then create a stable micro-chimera of the recipient.
  • Recruiting the stem cells to the target site can be induced artificially by administering a suitable chemokine systemically or at the desired site via injection or through expression from the mesenchymal stem cells.
  • a suitable molecule is hypoxia inducible factor- 1, a chemokine such as stromal derived factor-1 (SDF-I).
  • Endothelial stem cells may also be recruited to the desired site by means of an interleukin, such as IL-I or IL- 8.
  • the cells may have the ability to replicate in certain drug screening and therapeutic applications, and to provide a reservoir for the generation of cardiomyocytes and their precursors.
  • the cells can optionally be telomerized to increase their replication potential, either before or after they progress to restricted developmental lineage cells or terminally differentiated cells. Stem cells that are telomerized may be taken down the differentiation pathway described earlier; or differentiated cells can be telomerized directly.
  • telomere catalytic component typically under a heterologous promoter that increases telomerase expression beyond what occurs under the endogenous promoter.
  • telomere particularly suitable is the catalytic component of human telomerase (hTERT), provided in International Patent Application WO 98/14592.
  • hTERT human telomerase
  • species homologs like mouse TERT WO99/27113
  • Transfection and expression of telomerase in human cells is described in Bodnar et al., Science 279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999.
  • hTERT clones (WO 98/14592) are used as a source of hTERT encoding sequence, and spliced into an EcoRl site of a PBBS212 vector under control of the MPSV promoter, or into the EcoRl site of commercially available pBABE retrovirus vector, under control of the LTR promoter.
  • Differentiated or undifferentiated stem cells are genetically altered using vector containing supernatants over a 8-16 h period, and then exchanged into growth medium for 1-2 days. Genetically altered cells are selected using a drug selection agent such as puromycin, G418, or blasticidin, and then recultured. They can then be assessed for hTERT expression by RT-PCR, telomerase activity (TRAP assay), immunocytochemical staining for hTERT, or replicative capacity.
  • TRAP assay telomerase activity
  • the following assay kits are available commercially for research purposes: TRAPezeTM, XL Telomerase Detection Kit (Cat. s7707; Intergen Co., Purchase N.Y.); and TeIoTAGGG Telomerase PCR ELISAplus (Cat.
  • TERT expression can also be evaluated at the mRNA by RT-PCR.
  • Available commercially for research purposes is the LightCycler TeIoTAGGG hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics).
  • Continuously replicating colonies are enriched by further culturing under conditions that support proliferation, and cells with desirable phenotypes can optionally be cloned by limiting dilution.
  • stem cells are differentiated into cardiomyocyte precursors, and then genetically altered to express TERT.
  • stem cells are genetically altered to express TERT, and then differentiated into cardiomyocyte precursors or terminally differentiated cells.
  • Successful modification to increase TERT expression can be determined by TRAP assay, or by determining whether the replicative capacity of the cells has improved.
  • telomeres are of particular interest in applications of where it is advantageous to have cells that can proliferate and maintain their karyotype— for example, in pharmaceutical screening, and in therapeutic protocols where differentiated cells are administered to an individual in order to augment cardiac function.
  • the cells can also be genetically altered in order to enhance their ability to be involved in tissue regeneration, or to deliver a therapeutic gene to a site of administration.
  • a vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type.
  • cells that are genetically altered to express one or more growth factors of various types cardiotropic factors such as atrial natriuretic factor, cripto, and cardiac transcription regulation factors, such as GATA-4, Nkx2.5, and MEF2-C. Production of these factors at the site of administration may facilitate adoption of the functional phenotype, enhance the beneficial effect of the administered cell, or increase proliferation or activity of host cells neighboring the treatment site.
  • transgenes into Stem Cells Means for introducing transgenes into cells are well known.
  • a variety of methods for delivering and expressing a nucleic acid within a mammalian cell are known to those of ordinary skill in the art. Such methods include, for example viral vectors, liposome-based gene delivery (WO 93/24640; Mannino Gould-Fogerite, BioTechniques 6(7):682-691 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987); and Budker et al, Nature Biotechnology, 14(6):760-764 (1996)).
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof. See, e.g., Buchscher et al, J. Virol. 66(5):2731-2739 (1992); Johann et al, J. Virol. 66(5):1635-1640 (1992); Sommerfelt et al, Virol. 176:58-59 (1990); Wilson et al, J. Virol. 63:2374-2378 (1989); Miller et al, J. Virol.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • AAV-based vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and polypeptides, and in vivo and ex vivo gene therapy procedures. See, West et al, Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO
  • Retroviral vectors are typically used for cells useful in the present invention.
  • Such vectors may comprise, for example, an HIV-2 packageable nucleic acid packaged in an HIV-2 particle, typically using a packaging cell line.
  • Cell transduction vectors have considerable commercial utility as a method of introducing genes into target cells.
  • gene therapy procedures in which the cell transduction vectors of the invention are used to transduce target cells with a therapeutic nucleic acid in an in vivo or ex vivo procedure may be used.
  • Stem cells such as CD34 + stem cells may be used in ex vivo procedures for cell transduction and gene therapy.
  • the present invention utilizes the feature that stem cells differentiate into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the heart for differentiation.
  • embodiments extend to directing stem cells to particular organs to regenerate tissue, such as to the heart to regenerate cardiac muscle cells.
  • Stem cells such a,s for example, pluripotent stem cells, can also be used in conjunction with the mesenchymal stem cells. If it is desired, the stem cells can also be differentiated in vitro.
  • Cells of this invention can be used to screen for factors (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of such cells and their various progeny.
  • factors such as solvents, small molecule drugs, peptides, oligonucleotides
  • environmental conditions such as culture conditions or manipulation
  • mesenchymal stem cells or other stem cell types are used to screen factors that promote maturation into later-stage cardiomyocyte precursors, or terminally differentiated cells, or to promote proliferation and maintenance of such cells in long-term culture.
  • candidate maturation factors or growth factors are tested by adding them to cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.
  • screening applications of this invention relate to the testing of pharmaceutical compounds for their effect on cardiac muscle tissue maintenance or repair. Screening may be done either because the compound is designed to have a pharmacological effect on the cells, or because a compound designed to have effects elsewhere may have unintended side effects on cells of this tissue type.
  • the screening can be conducted using any of the stem cells or terminally differentiated cells. The reader is referred generally to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat. No. 5,030,015.
  • Assessment of the activity of candidate pharmaceutical compounds generally involves combining the cells of this invention with the candidate compound, either alone or in combination with other drugs. The investigator determines any change in the morphology, marker phenotype, or functional activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlates the effect of the compound with the observed change.
  • Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and the expression of certain markers and receptors. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [ 3 H] -thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (pp 375-410 in In vitro Methods in Pharmaceutical Research, Academic Press, 1997) for further elaboration.
  • Effect of cell function can be assessed using any standard assay to observe phenotype or activity of cardiomyocytes, such as marker expression, receptor binding, contractile activity, or electrophysiology— either in cell culture or in vivo. Pharmaceutical candidates can also be tested for their effect on contractile activity—such as whether they increase or decrease the extent or frequency of contraction. Where an effect is observed, the concentration of the compound can be titrated to determine the median effective dose (ED50).
  • ED50 median effective dose
  • the amount of stem cells administered to the patient will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about IxIO 6 to about IxIO 12 , more preferably about IxIO 8 to about IxIO 11 , more preferably, about lxl0 9 to about IxIO 10 stem cells are utilized for adult humans. These amounts will vary depending on the age, weight, size, condition, sex of the patient, the type of tumor to be treated, the route of administration, whether the treatment is regional or systemic, and other factors. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient.
  • the present invention provides pharmaceutical compositions comprising mesenchymal stem cells.
  • pharmaceutical compositions comprise a mesenchymal stem cell and embryonic stem cells.
  • kits for treating cardiac tissue damage or for delivering a functional gene or gene product to the heart in a mammal comprising a mesenchymal stem cell.
  • Stem cells generally have been presented to the desired organs either by injection into the tissue, by infusion into the local circulation, or by mobilization of autologous stem cells from the marrow accompanied by prior removal of stem cell-entrapping organs before mobilization when feasible, i.e., splenectomy.
  • the administration of the stem cell compositions can be coupled with other therapies.
  • a therapeutic agent can be administered prior to, concomitantly with, or after infusing the stem cells to a patient.
  • Administration of cells transduced ex vivo can be by any of the routes normally used for introducing a cell or molecule into ultimate contact with blood or tissue cells.
  • the stem cells may be administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such cells in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Parenteral administration is one useful method of administration.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and in some embodiments, can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. These formulations may be administered with factors that mobilize the desired class of adult stem cells into the circulation.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by the vector as described above in the context of ex vivo therapy can also be administered parenterally as described above, except that lyophilization is not generally appropriate, since cells are destroyed by lyophilization.
  • the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular cells employed and the condition of the patient, as well as the body weight of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a cell type in a particular patient.
  • the physician should evaluate circulating plasma levels, and, in the case of replacement therapy, the production of the gene product of interest.
  • Transduced cells are prepared for reinfusion according to established methods. See, Abrahamsen et al, J. Clin. Apheresis 6:48-53(1991); Carter et al, J. Clin. Apheresis 4:113- 117(1988); Aebersold et al, J. Immunol. Methods 112: 1-7(1988); Muul et al, J. Immunol. Methods 101:171-181(1987) and Carter et al, Transfusion 27:362-365(1987). After a period of about 2-4 weeks in culture, the cells may number between IxIO 6 and IxIO 10 . In this regard, the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.
  • cells of the present invention can be administered at a rate determined by the LD 50 of the cell type, and the side effects of the cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
  • Adult stem cells may also be mobilized using exogenously administered factors that stimulate their production and egress from tissues or spaces, that may include, but are not restricted to, bone marrow or adipose tissues.
  • Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented.
  • Swine MSCs were isolated and expanded from a single, healthy male Yorkshire donor (Schuleri, K. H. et al. Am. J. Physiol Heart Circ. Physiol 294, H2002-H2011 (2008)). Briefly, bone marrow was obtained from the iliac crest, and aspirates were passed through a density gradient to eliminate undesired cell types and were plated with 25 ml MEM Alpha media (Mediatech, Manassas, VA) containing 20% fetal Bovine Serum (Hyclone, Logan, UT) in 162 cm 2 culture flasks (Fisher Scientific, Pittsburgh, PA).
  • MEM Alpha media Mediatech, Manassas, VA
  • fetal Bovine Serum Hyclone, Logan, UT
  • MSC population was then harvested and transduced with 5-Bromodeoxyuridin (BrdU) or green fluorescent protein (Lenti-GFP vector, Lentigen) according to manufacturers' instructions. All used cells were harvested when they reached 80-90% confluence at passage 5. Labeled MSCs were placed in a cryopreservation solution consisting of 10% DMSO, 5% porcine serum albumin, and 85% Plasmalyte.
  • Myocardial infarction was induced by accessing the Left Anterior Descending (LAD) coronary artery and occluding it after the first diagonal branch by inflating a coronary angioplasty balloon (2.75 x 15 mm). Because of differences in the coronary anatomy between Gottingen and Yorkshires, the balloon was inflated for 150min and 60min respectively in order to achieve comparable infarct sizes. Subsequently, the balloon was deflated, reperfusion was established and the carotid artery was permanently closed. All animals were adequately heparinized during the procedure.
  • LAD Left Anterior Descending
  • MI myocardial infarction
  • hypokinetic, akinetic, and dyskinetic areas were identified during contrast ventriculography, and injections were performed within and at the borders of the dysfunctional area, as defined by bi-plane ventriculography. A total of 15 injections were performed in each animal, with each injection containing 0.5 ml of the injectate. Each injection was fluoroscopic ally guided to distribute cells evenly throughout the entire infarct and border zones.
  • Cardiac MR imaging (CMR) of the Gottingen's heart function were performed at the following time points: baseline, 10 days, 1, 2 and 3 months post-MI and 1, 2 and 3 months following transplantation.
  • Serial CMR images were acquired with a four channel phase array, 1.5 T MR Scanner (Siemens Symphony, Er Weg, Germany) in anesthetized animals with electrocardiography gating and short breath-hold acquisition.
  • the protocol for cine- CMR images and tagging-CMR images has been described before (Amado, L. C. et al. J. Am. Coll. Cardiol. 48, 2116-2124 (2006); Amado, L. C. et al. Proc. Natl. Acad. Sci. U. S.
  • Regional strain magnitude was determined from the 24 radially displaced segments for each short axis section covering the entire LV and averaged among slices for each region and each time point, generating a strain map for each time point over the cardiac cycle.
  • the peak systolic circumferential strain (peak Ecc) was determined from the strain map for each time point. Delayed Contrast enhancement was used to assess the extension of the infarcted area.
  • C-kit ⁇ -sarcomeric actinin, ⁇ -smooth muscle actin, Connexin-43 (Sigma, Saint Louis, MO), N-cadherin, anti- GFP, Laminin, Phospho-Histone H3 (Abeam, Cambridge, MA), GATA-4, MDRl, Integrin- ⁇ l, CD3, CD14, CD68 (Santa Cruz Biotechnologies, Santa Cruz, CA), activated Caspase-3 (BD Biosciences, San Jose, CA), Nkx2.5 (R&D systems Inc, Minneapolis, MN), Factor VIII (Biocare Medical, Concord, CA) and KDR (Cell Signaling, Boston, MA).
  • FISH Fluorescence in situ Hybridization
  • tissues were digested for 3 min at 37 0 C with pepsin, washed with 2X SSC buffer (Invitrogen) and dehydrated through serial ethanol washing steps. The samples were air-dried and the probe was applied. After covering the samples with a coverslip and sealing them with rubber cement, the samples were placed in the hybridizer (Dako) for denaturation (lOmin at 8O 0 C) followed by overnight hybridization at 37 0 C. The next day, samples were washed with 2X SSC, mounted with DAPI and covered.
  • 2X SSC buffer Invitrogen
  • the numbers of GFP pos , c-kit pos and Y pos cells were quantitated per square millimeter (mm ). Apoptotic and mitotic cells at 24h and 72h time points were expressed as the percentage of the GFP pos cells co-localized with activated caspase 3 and phospho-H3 respectively. The same approach was followed for the quantification of the cardiac and vascular commitment of the allografts as well as the endogenous c-kit pos cells. Morphometric analysis was performed by using a custom research package (Image J, NIH, Bethesda, Maryland). For assessing the density of newly formed vessels, the axial ratio (major diameter/ minor diameter) for each Y chromosome containing vascular structure was obtained.
  • SPSS for Windows version 15.0 SPSS Inc., Chicago, IL.
  • Differences between groups following immunohistological evaluation were compared by using One Way ANOVA.
  • Differences between groups in ejection fraction and infarct size based on MR images were calculated by using one-way ANOVA within different time points.
  • the Tukey's test was used for the post-hoc analysis.
  • Difference in peak Ecc were calculated by repeated measurements 2 way analysis of variance with one factor repetition. Pearson correlation was applied to find a relation between cell engraftment and increase in peak Ecc. A level of P ⁇ 0.05 was considered statistically significant.
  • MI myocardial infarction
  • TEI transendocardial injections
  • ⁇ MSCs allogeneic adult bone marrow derived mesenchymal stem cells
  • Example 1 Mesenchymal stem cells regenerate cardiac muscle, vasculature and stem cell niches
  • GFP labeled male allogeneic adult bone marrow derived mesenchymal stem cells were injected into female pigs following myocardial infarction (MI) ( Figure 5).
  • MI myocardial infarction
  • ⁇ MSCs allogeneic adult bone marrow derived mesenchymal stem cells
  • Endogenous c-kit pos cardiac stem cells CSCs
  • Endogenous c-kit pos CSCs were rare 1-3 days after transplantation, but dramatically increased in the ⁇ MSCs treated pigs by 2 weeks ( Figures 2G, 2H).
  • the number of c-kit cells increased by 50-fold in the infarct zone of the treated animals and was also elevated, though less dramatically, in border and remote zones of MSC treated animals ( Figures 9A-9D).
  • These c- kit pos cells were CD3 neg , CD14 neg and CD68 neg , excluding inflammatory and mast cells phenotypes, and were detected in clusters in the infarcted and border zones of the ⁇ MSCs treated animals.
  • c-kit cells were observed as a rare isolated cell and were not part of cell clusters ( Figures 2G, 2H, and Figures 7C and Figures 9A-9D] .
  • Figures 2G, 2H, and Figures 7C and Figures 9A-9D To address whether endogenous c-kit cells were coupled to host or chimeric cells, the expression of connexin-43 and N-cadherin was tested; c-kit cells formed gap junctions and mechanical connections via these proteins with other c-kit cells as well as with GFP pos MSCs ( Figures 21, 2J). These cell-cell interactions closely resemble cardiac stem cell niches. Further immunohistochemical characterization demonstrated that a number of these cells co-expressed MDRl and GATA-4, indicating the commitment to cardiac lineage within the regenerated niches.
  • Intramyocardial injection of ⁇ MSCs three months after MI produced substantial increases in ejection fraction, reductions in scar, and restored regional cardiac function in the border zones of the scar corresponding to the regions of scar reduction ( Figures 3A-3H).
  • the reduction in scar size correlated closely with regional functional recovery ( Figure 3H).
  • the hearts were analyzed by immunohistologic examination to test whether this functional recovery required cell engraftment.
  • Y chromosome containing cells (Y pos ) were present in the infarct and border zones of the ⁇ MSC-treated group, but were not detected in the placebo group, nor in the remote zones of the ⁇ MSC-treated animals.
  • Y pos cells were also present in both large and small vascular structures, supporting participation in new vessel formation in IZ and BZ.
  • Donor cells differentiated into both vascular muscle and endothelial lineages, as evidenced by co-localization of ⁇ -smooth muscle actin and factor VIII, respectively [Fig 4 ac].
  • An overall of 9.9 + 2.4% of the Ypos cells were incorporated into vessel walls, with 5.9 + 1.9% co-expressing Factor VIII and 4.0 + 1.6% ⁇ -smooth muscle actin.
  • the density of newly-formed vessels was calculated in vessel length per cubic millimeter. In IZ, the density of the cells was 0.05+0.01 mm/ mm 3 and in the BZ it was similarly 0.04+ 0.02 mm/ mm 3 [Fig 4 d].
  • MSCs are a true stem cell population with known immunomodulatory and immuno-tolerogenic capacities that are a safe and successful cardiac allograft. MSCs meet all the expectations of a cell product that can be readily available in therapeutic quantities, thus providing an "off-the shelf cell-based therapy for treating heart disease. Together these findings demonstrate that allogeneic MSCs, unaltered to enhance their survival, engraft and exhibit trilineage cardiac cell differentiation. The repair process is driven by damage signals, and occurs both in acute and in chronic myocardial infarction.
  • MSC engraftment is accompanied by the formation of cell-cell interactions both with host adult cells and with host endogenous precursor cells.
  • the latter reconstitutes multi-cellular clusters that have the features of stem cell niches and promotes the migration and myocytic differentiation of endogenous cardiac precursor cells.
  • the degree of engraftment correlates with recovery of cardiac function, which is substantial, and with reduction in myocardial scar formation. While scar reduction is incomplete, these findings raise the possibility that strategies involving repeated ⁇ MSCs applications or acceleration of their differentiation rates could result in near total elimination of cardiac scar.

Abstract

L'invention concerne des compositions comprenant des cellules souches fournies dans un myocarde infarci par injection endocardiaque, qui se greffe et se différencie en myocytes, cellules endothéliales et muscle lisse vasculaire sans nécessiter de modifications destinées à améliorer la survie. Que les cellules soient injectée rapidement (des jours) ou tardivement (des mois) après l'infarctus du myocarde, la greffe prend ; l'efficacité de la prise de greffe est corrélée à la récupération fonctionnelle du cœur. Les cellules souches recrutent également des cellules précurseurs cardiaques endogènes, reconstituent des niches de cellules souches du myocarde et améliorent la différenciation de cellules endogènes en myocytes.
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